Butylamine

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Endpoint:

toxicity to microorganisms

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1976

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Acceptable, well-documented publication which meets basic scientific principles.

Qualifier:

equivalent or similar to guideline

Guideline:

ISO 10712 (Water quality – Pseudomonas putida growth inhibition test (Pseudomonas cell multiplication inhibition test

Principles of method if other than guideline:

Cell Multiplication Inhibition Test

GLP compliance:

no

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

TEST SOLUTION

Prior to preparation of dilution series, the test item stock solution of known concentration was neutralized to pH 7.0. In parallel, a dilution series for a second test with test item solutions of non-adjusted pH was prepared.
Four parallel dulution series were prepared in 300 ml Erlenmayer flasks, stoppered with cotton-lined plastic caps. Each of the dilution contained one part (v/v) of the pollutant solution in 2^0 to 2^14 parts (v/v) of the mixture. The dilution series was prepared as follows: The first flask of each solution series was filled with 160 ml test substance stock solution, which was prepared with double-distilled water. From the first flask 80 ml stock solution was transferred in the second flask and 80 ml double-distilled water was added. The dilution scheme was applied to the following flasks.

Three of the four parallel dilution series (one was kept as control) were inoculated with 10 ml adjusted bacterial suspension (see details on test inoculum); 5 ml of stock solution I and 5 ml of stock solution II were added (for stock solution I and II see any other additional information on materials and methods). Controls were filled with 10 ml saline, 5 ml stock solution I and 5 ml stock solution II (for saline see any other additional information on materials and methods). The final volume of all flasks was 100 ml.

Test organisms (species):

Pseudomonas putida

Details on inoculum:

TEST INOCULUM

Laboratory stock cultures of Pseudomonas putida were kept on the nutrient medium in agar slant tubes (for nutrient medium see any other additional information on materials and methods). For the cell multiplication inhibition test, stock cultures of Pseudomonas putida were incubated at 25 °C for 24 h. After 24 h, the cell material was washed off with sterile saline. The extinction value of monochromatic radiation at 436 nm for a 10 mm layer was photoelectrically measured. By onward dilution with saline the extinction value was finally adjusted to a turbidity corresponding to the extinction value of a Formazin standard suspension TE/F/436 nm = 10. The adjusted bacterial solution was used as test inoculum.

Details on cultivation of stock cultures:
Stock cultures and preliminary cultures were kept on the same nutrient in agar slant tubes (change interval: 1 week). Inoculated stock cultures are incubated at 25 °C for 24 h.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

16 h

Test temperature:

25°C

pH:

7.0 (neutralised)

Nominal and measured concentrations:

no data

Details on test conditions:

TEST PRINCIPLE
The concentration of bacterial suspensions was determined by photometric measurements and expressed by the extinction value of the primary light of monochromatic radiation at 436 nm for a layer of 10 mm thickness. The concentration at which an inhibitory effect of the test substance occurred (TTC = toxicity threshold concentration) was derived on the basis of a calculated extinction value that was 3% below the mean value of extinction for all non-toxic dilutions in the test.

TEST SYSTEM
- Test vessel: 300-mL Erlenmeyer flask
- Type (delete if not applicable): open
- Fill volume: 100 mL
- Aeration: no, by shaking
- No. of organisms per vessel: cell density as extinction at 436 nm: the extinction value of the inoculum solution (see section "Details on inoculum") was finally adjusted to a turbidity corresponding to the extinction value of a Formazin standard suspension TE/F/436 nm = 10. 10 mL of this adjusted bacterial suspension was used as test inoculum.
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 1

TEST PROCEDURE
The inoculated dilution series and the controls were incubated for 16 hours at 25°C. Afterwards, the extinction of monochromatic radiation at 436 nm in a 10 mm layer was measured for the inoculated dilution series. In case discolouration or turbidity occurred, respective controls were used as photometric blank value.

EVALUATION
Graphical evaluation of effect concentration were performed after the test using means of measured extinction values, i.e. the extinction mean of all non-toxic test solutions A (SD of < 3%) and the mean of the lowest observed effect concentration B. A horizontal line was plotted for the mean extinction value A and B on a semi-logarithmic scale. The coordinate of the highest observed non-toxic concentration P-NOEC and lowest observed toxic concentration P-LOEC were marked on the respective lines. A regression line was drawn from P-NOEC to P-LOEC. In order to determine the TTC (toxicity threshold concentration, adverse effect of ca. 3%) a horizontal line was plotted at the extinction value A-3%. From the intersection point between the horizontal line A-3% and the regression line from P-NOEC to P-LOEC the TTC was derived.

OTHER TEST CONDITIONS
- Adjustment of pH: first test no, second test yes
- Photoperiod: no data
- Light intensity: no data

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- growth (cell multiplication; determined turbidimetrically)

Key result

Duration:

16 h

Dose descriptor:

other: TTC

Effect conc.:

800 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth inhibition

Remarks on result:

other: TTC = toxicity threshold concentration (approx. EC3)

Remarks:

stock solution neutralized to pH 7

Duration:

16 h

Dose descriptor:

other: TTC

Effect conc.:

65 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth inhibition

Remarks on result:

other: TTC = toxicity threshold concentration (approx. EC3)

Remarks:

pH not neutralized

Validity criteria fulfilled:

not applicable

Remarks:

no guideline followed

Conclusions:

The TTC (=EC3)-value of 800 mg/L (Pseudomonas putida; 16 h; growth inhibition) does not indicate substantial toxicity to microorganisms.

Executive summary:

The effect of n-butylamine solutions (two tests performed: with non-neutralized test item solutions as well as with stock solution neutralized to pH 7.0) on Pseudomonas putida was tested in a cell multiplication inhibition test over a test period of 16 hours. The concentration of bacterial suspensions was determined by photometric measurements and expressed by the extinction value of the primary light of monochromatic radiation at 436 nm for a layer of 10 mm thickness. The concentration at which an inhibitory effect of the test substance occurred (TTC = toxicity threshold concentration) was derived on the basis of a calculated extinction value that was 3% below the mean value of extinction for all non-toxic dilutions in the performed test. The derived TTC (16 h) for the test substance was 800 mg/L (neutralized) and 65 mg/L (non-neutralized). It must be assumed that sewage is neutralized to an acceptable pH range before discharge into STP. Further, sewage will have a buffer capacity on its own such that extreme pH values will not occur. Therefore, the TTC of 800 mg/L is the relevant value to be used in hazard and risk assessment for STP microorganisms. In conclusion, any inhibition of the degradation activity of activated sludge microorganisms by n-butylamine is not anticipated when introduced in concentrations below this value.

Description of key information

TTC (=EC3)-value (Pseudomonas putida; 16 h; growth inhibition) =  800 mg/L (study performed similar to ISO-10712)

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

800 mg/L

Additional information

The effect of n-butylamine solutions (two tests performed: with non-neutralized test item solutions as well as with stock solution neutralized to pH 7.0) on Pseudomonas putida was tested in a cell multiplication inhibition test over a test period of 16 hours (study performed similar to ISO-10712). The concentration of bacterial suspensions was determined by photometric measurements and expressed by the extinction value of the primary light of monochromatic radiation at 436 nm for a layer of 10 mm thickness. The concentration at which an inhibitory effect of the test substance occurred (TTC = toxicity threshold concentration) was derived on the basis of a calculated extinction value that was 3% below the mean value of extinction for all non-toxic dilutions in the performed test. The derived TTC (16 h) for the test substance was 800 mg/L (neutralized) and 65 mg/L (non-neutralized). It must be assumed that sewage is neutralized to an acceptable pH range before discharge into STP. Further, sewage will have a buffer capacity on its own such that extreme pH values will not occur. Therefore, the TTC of 800 mg/L is the relevant value to be used in hazard and risk assessment for STP microorganisms. In conclusion, any inhibition of the degradation activity of activated sludge microorganisms by n-butylamine is not anticipated when introduced in concentrations below this value.

In support of this value is the Zahn-Wellens test on inherent biodegradability (1977/1980). The initial concentration applied was 1000 mg/L COD (neutralized), and very rapid biodegradation was observed (90 % within 2 days, 100% within 5 days). This allows to exclude any relevant inoculum toxicity at this concentration.

In a 7-d toxicity pre-test preceding the main biodegradation study, 100 mg n-butylamine/L failed to inhibit the metabolism of the inoculum (here: activated sludge), indicated by highly efficient metabolic oxidation of glucose and n-butylamine itself (Yoshimura et al. 1980: see Section 5.2.1). A higher oxygen consumption was observed with glucose plus n-butylamine compared to glucose alone.

The other three pioneer studies referred to are based on pure cultures of non-standard test organisms (protozoae). All three organisms are of low relevance for the STP process (organisms feeding on bacteria). No standardized testing protocols do exist and thus reliability of the study outcome is not assessable. In conclusion, these are unsuitable test systems, and studies on Entosiphon and Chilomonas are therefore disregarded  [Bringmann and Kühn 1978 - 1982].